Summary: Protection against invasive disease by the pneumococcus is mediated by anti-polysaccharide (PS) antibodies that are opsonic. The recently licensed pneumococcal conjugate vaccine, Prevnar, contains PS from the seven most prevalent pneumococcal types, 4, 6B, 9V, 14, 18C, 19F,and 23F, conjugated to the diphtheria CRM197 protein. The serotype 19F conjugate induced higher antibody levels in clinical studies than several of the other serotypes, yet was isolated from a high percentage of otitis media vaccine failures. We have therefore investigated possible alterations in the epitopes expressed by 19F conjugates prepared using reductive amination. It is important to know whether chemical modifications used to activate and then conjugate pneumococcal PSs to protein carriers result in modifications to any of the PS epitopes. Since the immune response to PS antigens is clonally restricted, we screened human sera by ELISA for antibodies to 19F using the native (unmodified) PS, 19F conjugated to tyramine using a cyanylation method, and 19F conjugated to human serum albumin (HSA) by reductive amination. We examined 61 normal adult sera obtained from the NIH blood bank, 24 pairs of pre and post pneumococcal PS vaccination sera, 10 pairs of sera pre and post Merck pneumococcal conjugate vaccination, and 10 pairs of sera pre and post Lederle pneumococcal conjugate vaccination. We also used monoclonal antibodies induced by a 19F conjugate. Two out of the 85 normal sera were found to have highly reactive antibodies to the 19F-HSA antigen, but were non-reactive to the native 19F antigen. The source of the antibodies reactive with the PS by modified by reductive amination was investigated using competitive inhibition using PS from the other serotypes in group 19, 19A, 19B, and 19C, and some Klebsiella pneumoniae PSs known to cross-react with 19F. The 19F conjugate contained in Prevnar expressed more of the epitope than the 19F-HSA conjugate prepared in our lab. We compared post Prevnar 19F conjugate vaccination sera to post Merck conjugate (used another conjugation method) vaccination sera. Only the Prevnar conjugate induced antibodies to the modified epitope. The monoclonal antibodies showed equal binding to both the native and modified Pn 19F antigen preparations. Functional activity of these antibodies to the different 19F epitopes will be determined by opsonophagocytosis.